Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths in the world with over 1 million cases annually and a median survival time of only 7.8 months in untreated patients. Conditionally replicating viruses targeted to tumors are being developed as a novel class of oncolytic agents for cancer treatment. Vesicular Stomatitis Virus (VSV) is a RNA virus with inherent specificity for replication in tumor cells due to their substantially attenuated anti-viral responses. We demonstrated robust VSV replication and cytopathic effects in cultured rat and human HCC cells, while normal rat and human hepatocytes were completely refractory. We have also constructed a recombinant VSV that expresses a genetically modified fusogenic membrane glycoprotein of the Newcastle disease virus (rVSV-F) and showed that through hepatic artery infusion, it infected and replicated extensively in multi-focal HCC lesions pre-established in the livers of syngeneic and immune-competent rats. The treatment resulted in massive tumor destruction and substantial survival prolongation, and without liver pathology, in the animals. To clinically translate these exciting proof-of-principle laboratory findings, we propose to perform a series of preclinical pharmacological and toxicological studies to assess the safety of rVSV-F in support of an IND to conduct a Phase I clinical translational trial in advanced HCC patients, which will include VSV replication in normal human cells in vitro and assessment of acute toxicity in tumor-bearing rats. Long-term toxicity will be assessed in normal rats and rhesus monkeys. We will also produce, purify, and fully characterize a clinical lot of rVSV-F under cGMP for use in the Phase I dose escalation trial in HCC patients, who will be limited initially to those with Child-Pugh A liver functions and without viral hepatitis. Finally, we will determine the effect of VSV on HCC lesions and surrounding liver tissues in surgically resected specimens from HCC patients of various etiologies including viral hepatitis. The respective tissue slices will be examined for cytopathic effects as well as replication of VSV and the hepatitis viruses in tumor versus liver cells, and the results will be used in considering the expansion of trial subjects to include those with certain underlying liver diseases. The successful conduct of the proposed clinical translational research may lead to the development of recombinant VSV vectors as a novel class of effective and safe oncolytic agents to treat patients with advanced HCC and other cancers in the future.